This invention relates to methods of inactivating microorganisms and manufacturing vaccines, to vaccines manufactured by such methods, and to kits incorporating such inactivated microorganisms. More particularly but not exclusively, this invention relates to methods of inactivating viruses and manufacturing virus vaccines and to vaccines manufactured from such methods.
Virus inactivation and safety tests are the most critical steps in the production of inactivated vaccines, in the case of foot-and-mouth disease (FMD) vaccines in particular, guaranteed safety is essential because any occurrence of the disease will cause a blockade of all export trade of animals and animal products.
A classical method of inactivating FMD virus and preparing FMD vaccine is with formaldehyde as described by Waldmann et al.: Waldmann O, Pyl G, Hobohm K O, Mohlmann H: xe2x80x9cDie Entwicklung des Riemser Adsorbatimpfstoffes gegen Maulxe2x80x94und Klauenseuche und seine Herstellungxe2x80x9d. Zbl Bakt I Orig 148: 1, 1941.
In the past this method was used in the preparation of FMD vaccines and e.g. applied in the preparation of vaccine based on Frenkel cultures; Frenkel S: xe2x80x9cModifications de la mxc3xa9thode de culture du virus aphteuse selon Frenkel. Valeur des vaccins selon les donnxc3xa9es du laboratoirexe2x80x9d. Bull OIE 1: 985, 1964.
Formaldehyde is known for its cross-linking action (fixation) of proteins, which probably caused the high stability and long shelf livesxe2x80x94of up to 5 years or longerxe2x80x94of FA-inactivated (Frenkel) vaccines.
Several studies showed that at the FA concentration prescribed by Waldmann, inactivation plots were not linear and often showed xe2x80x9ctailing offxe2x80x9d, which may cause incomplete inactivation. This was demonstrated in the following publications:
Graves J H: xe2x80x9cFormaldehyde inactivation of foot-and-mouth disease virus as applied to vaccine preparationxe2x80x9d, Am J Vet Res 24, 1131;
Weslen, T. and Dinter, Z.: xe2x80x9cThe inactivation of foot-and-mouth disease virus by formalinxe2x80x9d. Arch. Ges. Virusforsch. 1957, 7, 394;
Barteling S J, Woortmeijer R, Visser N: xe2x80x9cInnocuity testing of foot-and-mouth disease vaccines. I. Formaldehyde-inactivated alhydrogel vaccinesxe2x80x9d, J Biol. Stand. 1983, 11, 297.
Formaldehyde-inactivated vaccines induced good immunity and it was shown that under well-defined conditions linear inactivation plots can be obtained with FA (Barteling S J, Woortmeijer R: xe2x80x9cFormaldehyde inactivation of foot-and-mouth disease virus. Conditions for the preparation of safe vaccinexe2x80x9d, Arch Virol. 19B4, 80, 103). However, inactivation with FA remained suspicious and inactivation by aziridins (e.g. acetyl-ethylenimine, AEI) which more rapidly inactivates, with linear inactivation plots, became the method of choice.
Inactivation with AEI is described in the following publication:
Brown F, Hyslop NSG, Crick J, Morrow A W: xe2x80x9cThe use of acetyl-ethyleneimine in the production of inactivated foot-and-mouth disease vaccinesxe2x80x9d, J Hyg (Camb) 1963b, 61: 337.
A widely used method for inactivating FMD virus by an aziridin is described by H. G. Bahnemann: xe2x80x9cInactivation of viral antigens for vaccine preparation with particular reference to the application of binary ethyleniminexe2x80x9d. Vaccine, 1999, 8, 299.
However, unlike FA, aziridins have no cross-linking reactivity and vaccines prepared from some unstable FMD (vaccine) strains showed short shelf lives. Therefore, some labile vaccine strains are first fixed with FA before inactivation is completed with an aziridin compound such as BEI. This method has been described in the following 3 publications:
Rowlands et al. (1972)xe2x80x94Stabilizing the immunizing antigen of foot-and-mouth disease virus by fixation with formaldehyde. Arch. Ges. Virusforsch. 39, 274-283;
Mowat et al. (1973)xe2x80x94Enhancement of immunizing potency of foot-and-mouth disease vaccine for cattle by treatment of the antigen with formaldehyde. Arch.ges.Virusforsch. 41, 365-370; and
M M Rweyemamu et al. (1989)xe2x80x94Effect of formaldehyde (FA) and binary ethyleneimine (BEI) on the integrity of foot-and-mouth disease virus capsid. Rev. sci. tech. Off. Int. Epiz. 8, 747-767.
It is also known to inactivate the virus first with BEI and thereafter to cross-link or fix the inactivated virus particles or antigens with FA e.g. FMD virus antigen of the SAT2 Zim 7/83 strain.
A disadvantage of the known methods is that it takes relatively long (+/xe2x88x922 days) to obtain satisfactory levels of inactivation, with the result that part of the virus antigen may degrade. Vaccines manufactured from such inactivated virus antigens are therefore relatively less effective, in use.
Furthermore, BEI does not always give satisfying inactivation. In practice, the extrapolated results sometimes indicate that there are still chances of the presence of surviving virus entities in a batch at the end of the inactivation (at 48 hours). This means that such a batch is not acceptable and must be destroyed.
Objects of the present invention are to provide methods of inactivating microorganisms and manufacturing vaccines, to provide vaccines manufactured by such methods and to provide kits incorporating such inactivated microorganisms, which are improvements of the known methods, vaccines and kits.
According to a first aspect of the invention there is provided a method of inactivating a microorganism including the step of applying to said microorganism a cross-linking agent simultaneously with a separate inactivant.
The applicants have surprisingly found that by using the cross-linking agent simultaneously with the inactivant, a synergistic or augmenting effect is achieved whereby the microorganisms are inactivated, not only more thoroughly but, also, in a relatively much shorter period in comparison with the prior art methods.
The Applicants have further found that vaccines manufactured according to the invention are effective in use, probably due to the fact that the inactivation period is relatively shorter and that the cross-linking agent fixes the antigens of the microorganism, as they become available. The Applicants believe that the antigens are thus preserved and not degraded and, therefore, vaccines manufactured according to the invention are expected to be more stable with longer shelf lives than the known vaccines. Also, the Applicants further believe that vaccines will become less dependent on strict cold-chain conditions during transport and use in the field.
The cross-linking agent may comprise an aldehyde.
The aldehyde may be a di-aldehyde.
Preferably the aldehyde is formaldehyde (FA).
The Applicants further believe that, at least in the case of FA, the cross-linking agent prepares the virus for inactivation by the inactivant.
The inactivant may be an aziridine compound. The aziridine compound may be an ethyleneimine.
Preferably the ethyleneimine is binary ethyleneimine (BEI).
The method may include the further step of stopping the inactivation process.
The inactivation process may be stopped by the addition of any one or more of sodium thiosulphate, sodium bisulfite, and trishydroxymethyl-aminomethane (Tris-buffer).
According to a second aspect of the invention there is provided a vaccine including a microorganism inactivated by the above method according to the first aspect of the invention.
According to a third aspect of the invention there is provided a method of manufacturing a vaccine including the steps of:
providing a pathogen;
inactivating the pathogen by applying to said pathogen a cross-linking agent simultaneously with a separate inactivant;
the cross-linking agent may also inactivate the pathogen (by its cross-linking activity).
The cross-linking agent may comprise an aldehyde.
The aldehyde may be a dialdehyde.
Preferably the aldehyde is formaldehyde (FA).
The inactivant may be an aziridine compound. The aziridine compound may be an ethyleneimine.
Preferably the ethyleneimine is binary ethyleneimine (BEI).
The method may include the further step of stopping the inactivation process.
The inactivation process may be stopped by the addition of any one or more of sodium thiosulphate, sodium bisulfite, and trishydroxymethyl-aminomethane (Tris-buffer).
The Applicants have further found that the above methods according to the invention are particularly effective in the inactivation of viruses and the preparation of vaccines for viral pathogens.
The virus may be of the family picoma viridae.
Preferably the virus is a foot-and-mouth disease virus, but it will be appreciated that the methods according to the invention will be effective in respect of most viruses and even other microorganisms.
According to another aspect of the invention there is provided a test kit including a microorganism inactivated by the above method of the invention. The inactivated microorganism may be in the form of an allergen or antigen for use in an ELISA.